Detection of colorectal cancer (CRC) by examination of peripheral blood samples could enhance screening efforts of the general population and lead to better survival rates in those patients with this type of cancer. Patients have a much higher survival rate when CRC is detected at an early stage. Furthermore, even within the population of patients diagnosed with a specific stage of CRC, there can be dramatic differences in prognosis for reasons that are understood poorly. Rapid developments in proteomic analytical approaches suggest the potential to identify candidate CRC-selective biomarker proteins, akin to Prostate Specific Antigen (PSA) for prostate cancer or CA125 for ovarian cancer. A rational approach to identifying an appropriate CRC biomarker panel would be to first determine what proteins are uniquely present or elevated in colon cancer cells, compared to normal colon cells, or shed into the interstitial fluid around the tumor cells. Having identified CRC-selective proteins, one could then investigate which may be elevated in the peripheral blood. This strategy for comprehensive and detailed comparative analysis of CRC vs. normal intestinal epithelial cells could overcome key hindrances that beset many blood-screening approaches, which are often confounded by the fact that just a handful of high-abundance proteins represent more than 90% of the blood protein content. For most analytical approaches, these high abundance proteins provide a fog that obscures CRC-selective proteins or peptides. Here we propose a strategy and workflow that incorporate several interrelated analytical advances in proteomic sample processing, nano-scale liquid chromatography (nano-LC), and mass spectrometry (MS) that enhance the suitability of nano-LC/MS approaches for the analysis of tissues. These advances provide (i) highly quantitative recovery of tissue proteins (ii) highly reproducible and sensitive protein expression profiling, (iii) quantification of a significantly greater number of proteins than otherwise feasible, and (iv) highly sensitive quantification of specific proteins of interest. Employing this analytical strategy, we will (1) perform comparative proteomic analysis of cells derived from CRC vs. adjacent normal colon epithelium, in order to identify CRC-selective proteins, (2) investigate the complement of proteins or peptides that CRC cells may secrete into the extracellular environment, as these may have higher likelihood of making their way to peripheral blood, and (3) evaluating strategies to quantify CRC-selective proteins in the peripheral blood, using patient-matched blood samples collected prior to cancer surgery. Establishing the potential of this workflow and strategy would be essential for the logical follow-on to the proposed project, which would be to seek CRC-selective proteins in peripheral blood in a comprehensive clinical study. The overall outcomes of the proposed study include a blood based screening tool for the early detection of CRC, as well as a more comprehensive understanding of CRC-selective markers that could improve assessment of patient prognosis, or stratification of disease for more effective therapy.